Method and apparatus for washing solid substrate with ultrasonic wave after hybridization reaction

ABSTRACT

A method and apparatus for washing solid substrate with ultrasonic wave after hybridization reaction are provided. The method includes (1) putting the solid substrate into a container having a washing solution, on which the hybridization reaction has been completed, (2) using an ultrasonic generator to produce a certain strength of ultrasonic wave and transmit it into the washing solution, (3) washing the substrate by means of the cavitation effect being produced in the washing solution by the ultrasonic wave. Since the cavitation bubbles in the washing solution can produce intensive efflux and local microslipstream against the solid surface, evidently reducing the liquid surface tension and friction and enhance the liquid flow, non-specific adsorbate and deposit bound weakly on the solid substrate can be washed down rapidly.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application of PCT applicationPCT/CN2002/000877, filed on Dec. 10, 2002, which claims priority toChinese patent application Serial No. 02128860.7, filed Aug. 16, 2002.The contents of the above referenced applications are incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a method and device for washing solidsubstrate after hybridization reaction using ultrasonic wave, especiallyrelates to the method and device for washing biochips afterhybridization reaction. The method and device can be used in clinicaldiagnosis, basic life science research, agriculture, environmentalmonitoring, and other fields.

BACKGROUND OF THE INVENTION

Nonspecific adsorption is very common during hybridization reactions,which makes it difficult to analyze the hybridization results inbiological and medical applications. Thus, washing to get rid ofnonspecific adsorption is one of the important steps during biologicaland medical sample analysis and detection. The conventional washingmethod is carried out by submerging the surface area of the solidsubstrate to be washed in a washing solution and washing the solidsubstrate through mechanical vibration. Although such method is simple,it is time consuming and inefficient, leaving high background signalsand poor uniformity. Furthermore, because such washing device has movingpart, it is difficult to adapt the device for integration andminiaturization.

BRIEF SUMMARY OF THE INVENTION

The purpose of the present invention is to shorten the washing timeafter hybridization reaction, increase the contrast between thehybridization and background signals, and provide a hybridizationreaction washing device that is structurally simple, fast, inexpensive,easy to operate, suitable for automation, and applicable tomicroreaction systems.

The purpose of the present invention is achieved by the followingtechnical scheme: a method for washing a hybridized solid substrate(i.e., a solid substrate on which a hybridization reaction has beencompleted) using ultrasonic wave, the method includes 1) placing thehybridized solid substrate in a container having a washing solution; 2)generating a certain strength of ultrasound with an ultrasonic wavegenerator and transmitting the ultrasound into the washing solution,wherein the washing of the solid substrate is facilitated by cavitationeffects in the washing solution produced by the ultrasound.

The present invention further provides a device for carrying out thewashing method described above. The device includes a container having awashing solution, a hybridized solid substrate placed in the washingsolution, wherein at least one ultrasonic resonance component is placedupon, below, or around the hybridized solid substrate, and wherein theultrasonic resonance component is connected to a resonance circuit.

When washing, first place the hybridized solid substrate in the washingsolution, then activate the ultrasonic resonance circuit, which drivesthe ultrasonic resonance component to produce ultrasound. The washing isfacilitated by the cavitation effect produced by the ultrasoundtransmitted to the solution. The cavitation bubbles in the washingsolution can produce violent jet streams and local microslipstreamagainst the solid surface, evidently reduce the liquid surface tensionand friction, destroy the boundary layer at the solid-liquid interface,and enhance the liquid flow. Accordingly, nonspecific adsorption anddeposit bound weakly on the solid substrate can be rapidly washed away.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an exemplary device of the presentinvention.

FIG. 2 is the front view of an ultrasonic resonance component made ofpiezoelectric ceramic disk used in an example of the present invention.

FIG. 3 is the back view of an ultrasonic resonance component made ofpiezoelectric ceramic disk used in an example of the present invention.

FIG. 4 shows the comparison of hybridization fluorescent signalsobtained according to an exemplary method of the present invention andthose obtained according to the conventional mechanical vibrationmethod.

FIG. 5 shows the comparison of background fluorescent signals obtainedaccording to an exemplary method of the present invention and thoseobtained according to the conventional mechanical vibration method.

DETAILED DESCRIPTION OF THE INVENTION

Some embodiments of the present invention are hereinafter described withreference to the drawings.

In some embodiments, the hybridized solid substrate is first placed inthe washing solution, wherein the surface of the substrate that is to bewashed is brought into contact with the washing solution. The ultrasonicresonance component can be in direct contact with the washing solution,or can be adhered to the outside wall of the solution container byadhesives such as epoxy adhesives. The ultrasonic resonance componentcan be positioned at the upper part, the lower part, or the peripheralof the container. The material for the ultrasonic resonance componentcan be piezoelectric ceramic or quartz material. The shape of theultrasonic resonance component can be column, cone, cylinder, cube, orany other shape suitable for generating ultrasonic wave resonance. Thesize of the ultrasonic resonance component can be in a range of 10 μm³to 1000 cm³. The frequency of the ultrasonic wave generated by theultrasonic resonance component can be in a range of 20 kHz to 100 MHz.The power of the ultrasonic wave generator can be in a range of 1 W to200 W, and the mode of generating ultrasonic wave can be continuous orintermittent.

FIG. 1 shows an actual example according to the present invention. Thisexample provides a method of washing a hybridized gene chip usingultrasonic wave. A round piezoelectric ceramic disk 2 with diameter of2.5 cm and frequency of 1.7 MHz was adhered to the inner side of plasticbox 1 with the dimension of 8.5 cm×5 cm×2 cm. The piezoelectric ceramicdisk 2 was connected to ultrasonic resonance circuit 5 by wire 3 passingthrough a hole drilled at the side wall of the plastic box 1. The outputpower of ultrasonic resonance circuit 5 was 20 W. The hole for wirepassing was then sealed with epoxy adhesive. The outside rim ofpiezoelectric ceramic disk 2 was encircled by circular rubber sealingloop 4. The rubber sealing loop 4 was affixed to the bottom of plasticbox 1 by epoxy adhesive 7, which was used to insulate the solder pointfrom the washing solution to avoid short circuit. When washing, the genechip to be washed 6 was submerged into washing solution 8 in plastic box1.

FIGS. 2 and 3 show the arrangement of electrodes on the piezoelectricceramic ultrasonic resonance component of the present invention. FIG. 2is the front view of the piezoelectric ceramic disk, and FIG. 3 is theback view of the same: 9 represents the metal electrode coated on thesurface of the ultrasonic piezoelectric ceramic disk, 3 represents thewire soldered to the ultrasonic piezoelectric ceramic disk, and 10represents the exposed piezoelectric material at the back of theultrasonic piezoelectric ceramic disk.

FIGS. 4 and 5 show the comparison of hybridization and backgroundfluorescent signals obtained according to an exemplary method of thepresent invention and those obtained according to the conventionalmethod. Washing condition 1 shown in FIGS. 4 and 5 represents washingthe liquid-solid hybridization system for 15 minutes by mechanicalvibration (vibration frequency 80 rpm, amplitude 15 mm). Washingcondition 2 represents washing the same system for 3 minutes using a 1.7MHz piezoelectric ceramic disk under the power of 20 W. During washing,the gene chip was placed somewhere about 2 mm above the piezoelectricceramic disk, with its surface with probes on facing away from theceramic disk. The washing solution was then added to submerge the genechip. The washing solution was 0.1% SDS, and its volume was 10 mL.

The probe, hybridization sample, and hybridization condition for Array 1and Array 2 were the same. The probe was a 35mer oligonucleotide, whichwas attached covalently to an aldehyde modified glass slide. The targetsample was a 1045 bp DNA, part of which matches perfectly with theprobe. The sample DNA chain was labeled with Cy5 fluorescent molecules.The hybridization was carried out at 65° C. for 90 minutes. Thedetection was carried out using laser-induced fluorescence detectionmethod. The instrument for detection was ScanArray 4000. Thephotomultiplier tube (PMT) was set at 75% of the maximum setting of theinstrument. The power of the laser source was set at 80% of the maximumsetting of the instrument.

As shown in FIGS. 4 and 5, the 3-minute wash with the help of ultrasoundachieved the same washing results as the conventional washing method.Neither the hybridization fluorescent signals (FIG. 4) nor thebackground fluorescent signals (FIG. 5) showed obvious differencebetween the two methods. The experiment was repeated 8-9 times. Theseexperiments demonstrate that the same washing results as the mechanicalwashing method can be achieved by utilizing the cavitation effect ofultrasound in a relatively shorter time. Accordingly, the detectionprocess was accelerated.

INDUSTRIAL APPLICATIONS

Compared with conventional washing techniques in hybridizationreactions, the method of the present invention significantly shortensthe washing time (the results of the example show that the washing timeof the hybridized gene chip using the ultrasonic wave method was aboutone fifth of the time needed when using the conventional mechanicalvibration method) and increases the contrast of the hybridization andbackground signals. Furthermore, the structure of the inventive deviceis simple (the core elements only include the ultrasonic resonancecomponent, the corresponding ultrasonic resonance circuit, and thewashing solution container) and has no moving parts, which makes iteasier for integration and automation.

1. A method for washing a solid substrate on which a hybridizationreaction has been completed using ultrasonic wave, including 1) placingthe solid substrate in a container having a washing solution; 2)generating certain strength of ultrasound with an ultrasonic wavegenerator and transmitting the ultrasound into the washing solution,wherein the washing of the solid substrate is facilitated by cavitationeffect in the washing solution produced by the ultrasound.
 2. The methodof claim 1, wherein the power of the ultrasonic wave generator is in arange of about 0.1 W to about 200 W.
 3. The method of claim 1, whereinthe ultrasonic wave frequency generated by the ultrasonic wave generatoris in a range of about 20 kHz to about 100 MHz.
 4. The method of claim1, wherein the mode of generating ultrasonic wave by the ultrasonic wavegenerator is continuous or intermittent.
 5. A device for washing ahybridized solid substrate according to the method of claim 1,comprising a container having a washing solution and a hybridized solidsubstrate placed in the solution, wherein at least one ultrasonicresonance component is placed upon, below, or around the hybridizedsolid substrate, and wherein the ultrasonic resonance component isconnected with a resonance circuit.
 6. The device of claim 6, whereinthe resonance component is in direct or indirect contact with thewashing solution.